SRM/MRM Assay for the tyrosine-protein kinase receptor UFO (AXL) protein

ABSTRACT

Peptides from the tyrosine-protein kinase receptor UFO protein (AXL) are provided that are particularly advantageous for quantifying the AXL protein directly in biological samples that have been fixed in formalin by the method of Selected Reaction Monitoring (SRM)/Multiple Reaction Monitoring (MRM) mass spectrometry. Such biological samples are chemically preserved and fixed and include formalin-fixed tissue/cells, formalin-fixed/paraffin embedded (FFPE) tissue/cells, FFPE tissue blocks and cells from those blocks. A protein digest is prepared from the biological sample and the AXL protein is quantitated in the Liquid Tissue sample by the method of SRM/MRM mass spectrometry by quantitating in the protein sample at least one or more of the peptides described.

Cancer is treated with a collection of therapeutic agents that killgrowing and dividing cells and that function in a variety of ways. Acommon collection of chemotherapeutic agents has been used for decades,either individually or in combinations, and this common collection ofagents has become the traditional and routine cancer treatment inclinical oncology practice. These traditional chemotherapeutics agentsact by killing all cells that divide rapidly, one of the main propertiesof most cancer cells. However, these agents also kill growing normalcells and thus these agents are not considered to be “targeted”approaches to killing cancer cells. IN recent years a large group ofcancer therapeutic agents has been developed that target cancer cellswhere the therapeutic agent specifically attacks a protein that is onlyexpressed by the cancer cells and not by normal cells. This approach isconsidered to be a “targeted” approach to cancer therapy. Most recently,another approach to killing cancer cells in a targeted fashion has beento specifically modulate the immune system to enhance that ability ofthe cancer patient's immune system to kill cancer cells.

Therapeutic agents that target the tyrosine-protein kinase receptor UFOprotein, which can also be referred to as AXL, have shown promise inearly clinical trials. However, only those patients whose cancer cellsexpress high amounts of the AXL protein are likely to benefit fromtreatment with such AXL-targeted therapeutic agents. The methods belowprovide a quantitative proteomics-based assay that delivers a relevantmeasure of activation of the AXL signal pathway as AXL is not normallyexpressed in normal tissue and/or normal epithelial cells. Inparticular, the methods provide a mass spectrometry assay thatquantifies AXL in formalin fixed tissues from cancer patients and thatenables improved treatment decisions for cancer therapy.

Specific peptides derived from subsequences of the tyrosine-proteinkinase receptor UFO protein, also referred to as AXL, and be referred toherein as AXL, are provided. The peptide sequence andfragmentation/transition ions for each peptide are particularly usefulin a mass spectrometry-based Selected Reaction Monitoring (SRM), whichcan also be referred to as a Multiple Reaction Monitoring (MRM) assay,and will be referred to as SRM/MRM. The use of peptides for SRM/MRMquantitative analysis of the AXL protein is described.

This SRM/MRM assay can be used to measure relative or absolutequantitative levels of one or more of the specific peptides from the AXLprotein and therefore provide a means of measuring by mass spectrometrythe amount of the AXL protein in a given protein preparation obtainedfrom a biological sample.

More specifically, the SRM/MRM assay can measure these peptides directlyin complex protein lysate samples prepared from cells procured frompatient tissue samples, such as formalin fixed cancer patient tissue.Methods of preparing protein samples from formalin-fixed tissue aredescribed in U.S. Pat. No. 7,473,532, the contents of which are herebyincorporated by reference in their entirety. The methods described inU.S. Pat. No. 7,473,532 may conveniently be carried out using LiquidTissue reagents and protocol available from Expression Pathology Inc.(Rockville, Md.).

The most widely and advantageously available form of tissues from cancerpatients tissue is formalin fixed, paraffin embedded tissue.Formaldehyde/formalin fixation of surgically removed tissue is by farthe most common method of preserving cancer tissue samples worldwide andis the accepted convention for standard pathology practice. Aqueoussolutions of formaldehyde are referred to as formalin. “100%” formalinconsists of a saturated solution of formaldehyde (about 40% by volume or37% by mass) in water, with a small amount of stabilizer, usuallymethanol, to limit oxidation and degree of polymerization. The mostcommon way in which tissue is preserved is to soak whole tissue forextended periods of time (8 hours to 48 hours) in aqueous formaldehyde,commonly termed 10% neutral buffered formalin, followed by embedding thefixed whole tissue in paraffin wax for long term storage at roomtemperature. Thus molecular analytical methods to analyze formalin fixedcancer tissue will be the most accepted and heavily utilized methods foranalysis of cancer patient tissue.

Results from the SRM/MRM assay can be used to correlate accurate andprecise quantitative levels of the AXL protein within the specifictissue samples (e.g., cancer tissue sample) of the patient or subjectfrom whom the tissue (biological sample) was collected and preserved.This not only provides diagnostic information about the cancer, but alsopermits a physician or other medical professional to determineappropriate therapy for the patient. Such an assay that providesdiagnostically and therapeutically important information about levels ofprotein expression in a diseased tissue or other patient sample istermed a companion diagnostic assay. For example, such an assay can bedesigned to diagnose the stage or degree of a cancer and determine atherapeutic agent to which a patient is most likely to respond.

SUMMARY

The assays described herein measure relative or absolute levels ofspecific unmodified peptides from the AXL protein and also can measureabsolute or relative levels of specific modified peptides from the AXLprotein. Examples of modifications include phosphorylated amino acidresidues and glycosylated amino acid residues that are present on thepeptides.

Relative quantitative levels of the AXL protein are determined by theSRM/MRM methodology, for example by comparing SRM/MRM signature peakareas (e.g., signature peak area or integrated fragment ion intensity)of an individual AXL peptide in different samples. Alternatively, it ispossible to compare multiple SRM/MRM signature peak areas for multipleAXL signature peptides, where each peptide has its own specific SRM/MRMsignature peak, to determine the relative AXL protein content in onebiological sample with the AXL protein content in one or more additionalor different biological samples. In this way, the amount of a particularpeptide, or peptides, from the AXL protein, and therefore the amount ofthe AXL protein, is determined relative to the same AXL peptide, orpeptides, across 2 or more biological samples under the sameexperimental conditions. In addition, relative quantitation can bedetermined for a given peptide, or peptides, from the AXL protein withina single sample by comparing the signature peak area for that peptide bySRM/MRM methodology to the signature peak area for another and differentpeptide, or peptides, from a different protein, or proteins, within thesame protein preparation from the biological sample.

In this way, the amount of a particular peptide from the AXL protein,and therefore the amount of the AXL protein, is determined relative oneto another within the same sample. These approaches generatequantitation of an individual peptide, or peptides, from the AXL proteinto the amount of another peptide, or peptides, between samples andwithin samples, wherein the amounts as determined by signature peak areaare relative one to another, regardless of the absolute weight to volumeor weight to weight amounts of the AXL peptide in the proteinpreparation from the biological sample. Relative quantitative data aboutindividual signature peak areas between different samples are normalizedto the amount of protein analyzed per sample. Relative quantitation canbe performed across many peptides from multiple proteins and the AXLprotein simultaneously in a single sample and/or across many samples togain insight into relative protein amounts, such as one peptide/proteinwith respect to other peptides/proteins.

Absolute quantitative levels of the AXL protein are determined by, forexample, the SRM/MRM methodology whereby the SRM/MRM signature peak areaof an individual peptide from the AXL protein in one biological sampleis compared to the SRM/MRM signature peak area of a spiked internalstandard. In one embodiment, the internal standard is a syntheticversion of the same exact AXL peptide that contains one or more aminoacid residues labeled with one or more heavy isotopes. Such isotopelabeled internal standards are synthesized so that when analyzed by massspectrometry a standard generates a predictable and consistent SRM/MRMsignature peak that is different and distinct from the native AXLpeptide signature peak and which can be used as a comparator peak. Thuswhen the internal standard is spiked into a protein preparation from abiological sample in known amounts and analyzed by mass spectrometry,the SRM/MRM signature peak area of the native peptide is compared to theSRM/MRM signature peak area of the internal standard peptide, and thisnumerical comparison indicates either the absolute molarity and/orabsolute weight of the native peptide present in the original proteinpreparation from the biological sample. Absolute quantitative data forfragment peptides are displayed according to the amount of proteinanalyzed per sample. Absolute quantitation can be performed across manypeptides, and thus proteins, simultaneously in a single sample and/oracross many samples to gain insight into absolute protein amounts inindividual biological samples and in entire cohorts of individualsamples.

The SRM/MRM assay method can be used to aid diagnosis of the stage ofcancer, for example, directly in patient-derived tissue, such asformalin fixed tissue, and to aid in determining which therapeutic agentwould be most advantageous for use in treating that patient. Cancertissue that is removed from a patient either through surgery, such asfor therapeutic removal of partial or entire tumors, or through biopsyprocedures conducted to determine the presence or absence of suspecteddisease, is analyzed to determine whether or not a specific protein, orproteins, and which forms of proteins, are present in that patienttissue. Moreover, the expression level of a protein, or multipleproteins, can be determined and compared to a “normal” or referencelevel found in healthy tissue. Normal or reference levels of proteinsfound in healthy tissue may be derived from, for example, the relevanttissues of one or more individuals that do not have cancer.Alternatively, normal or reference levels may be obtained forindividuals with cancer by analysis of relevant tissues not affected bythe cancer. Assays of protein levels (e.g., AXL levels) can also be usedto diagnose the stage of cancer in a patient or subject diagnosed withcancer by employing the AXL levels. The level of an individual AXLpeptide is defined as the molar amount of the peptide determined by theSRM/MRM assay per total amount of protein lysate analyzed. Informationregarding AXL can thus be used to aid in determining the stage or gradeof a cancer by correlating the level of the AXL protein (or fragmentpeptides of the AXL protein) with levels observed in normal tissues.Once the quantitative amount of the AXL protein has been determined inthe cancer cells, that information can be matched to a list oftherapeutic agents (chemical and biological) developed to specificallytreat cancer tissue that is characterized by, for example, abnormalexpression of the protein or protein(s) (e.g., AXL) that were assayed.Matching information from an AXL protein assay to a list of therapeuticagents that specifically targets, for example, the AXL protein orcells/tissue expressing the protein, defines what has been termed apersonalized medicine approach to treating disease. The assay methodsdescribed herein form the foundation of a personalized medicine approachby using analysis of proteins from the patient's own tissue as a sourcefor diagnostic and treatment decisions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 (A-C) shows results of an SRM/MRM assay of a single peptide fromthe AXL protein performed on a positive control sample (not formalinfixed) and a Liquid Tissue lysate from a formalin fixed biologicalsample with quantitation of the AXL peptide conducted on atriplequadrupole mass spectrometer. The specific characteristics aboutthe precursor and transitions ions used to measure, using the SRM/MRMmethod, this peptide in biological samples that have been fixed informalin are shown.

DETAILED DESCRIPTION

In principle, any predicted peptide derived from the AXL protein,prepared for example by digesting with a protease of known specificity(e.g. trypsin), can be used as a surrogate reporter to determine theabundance of AXL protein in a sample using a mass spectrometry-basedSRM/MRM assay. Similarly, any predicted peptide sequence containing anamino acid residue at a site that is known to be potentially modified inthe AXL protein also might potentially be used to assay the extent ofmodification of the AXL protein in a sample.

AXL fragment peptides may be generated by a variety of means includingby the use of the Liquid Tissue protocol provided in U.S. Pat. No.7,473,532. The Liquid Tissue protocol and reagents are capable ofproducing peptide samples suitable for mass spectroscopic analysis fromformalin fixed paraffin embedded tissue by proteolytic digestion of theproteins in the tissue/biological sample. In the Liquid Tissue protocolthe tissue/biological is heated in a buffer for an extended period oftime (e.g., from about 80° C. to about 100° C. for a period of time fromabout 10 minutes to about 4 hours) to reverse or release proteincross-linking. The buffer employed is a neutral buffer, (e.g., aTris-based buffer, or a buffer containing a detergent). Following heattreatment the tissue/biological sample is treated with one or moreproteases, including but not limited to trypsin, chymotrypsin, pepsin,and endoproteinase Lys-C for a time sufficient to disrupt the tissue andcellular structure of said biological sample and to liquefy said sample(e.g., a period of time from 30 minutes to 24 hours at a temperaturefrom 37° C. to 65° C.). The result of the heating and proteolysis is aliquid, soluble, dilutable biomolecule lysate.

Surprisingly, it was found that many potential peptide sequences fromthe AXL protein are unsuitable or ineffective for use in massspectrometry-based SRM/MRM assays for reasons that are not immediatelyevident. As it was not possible to predict the most suitable peptidesfor MRM/SRM assay, it was necessary to experimentally identify modifiedand unmodified peptides in actual Liquid Tissue lysates to develop areliable and accurate SRM/MRM assay for the AXL protein. While notwishing to be bound by any theory, it is believed that some peptidesmight, for example, be difficult to detect by mass spectrometry becausethey do not ionize well or produce fragments distinct from otherproteins. Peptides may also fail to resolve well in separation (e.g.,liquid chromatography), or may adhere to glass or plastic ware.

AXL peptides found in various embodiments of this disclosure (e.g.,Tables 1 and 2) were derived from the AXL protein by protease digestionof all the proteins within a complex Liquid Tissue lysate prepared fromcells procured from formalin fixed cancer tissue. Unless notedotherwise, in each instance the protease was trypsin. The Liquid Tissuelysate was then analyzed by mass spectrometry to determine thosepeptides derived from the AXL protein that are detected and analyzed bymass spectrometry. Identification of a specific preferred subset ofpeptides for mass-spectrometric analysis is based on; 1) experimentaldetermination of which peptide or peptides from a protein ionize in massspectrometry analyses of Liquid Tissue lysates, and 2) the ability ofthe peptide to survive the protocol and experimental conditions used inpreparing a Liquid Tissue lysate. This latter property extends not onlyto the amino acid sequence of the peptide but also to the ability of amodified amino acid residue within a peptide to survive in modified formduring the sample preparation.

Protein lysates from cells procured directly from formalin(formaldehyde) fixed tissue were prepared using the Liquid Tissuereagents and protocol that entails collecting cells into a sample tubevia tissue microdissection followed by heating the cells in the LiquidTissue buffer for an extended period of time. Once the formalin-inducedcross linking has been negatively affected, the tissue/cells are thendigested to completion in a predictable manner using a protease, as forexample including but not limited to the protease trypsin. Each proteinlysate is turned into a collection of peptides by digestion of intactpolypeptides with the protease. Each Liquid Tissue lysate was analyzed(e.g., by ion trap mass spectrometry) to perform multiple globalproteomic surveys of the peptides where the data was presented asidentification of as many peptides as could be identified by massspectrometry from all cellular proteins present in each protein lysate.An ion trap mass spectrometer or another form of a mass spectrometerthat is capable of performing global profiling for identification of asmany peptides as possible from a single complex protein/peptide lysateis typically employed. Ion trap mass spectrometers however may be thebest type of mass spectrometer for conducting global profiling ofpeptides. Although an SRM/MRM assay can be developed and performed onany type of mass spectrometer, including a MALDI, ion trap, or triplequadrupole, the most advantageous instrument platform for an SRM/MRMassay is often considered to be a triple quadrupole instrument platform.

Once as many peptides as possible were identified in a single MSanalysis of a single lysate under the conditions employed, then thatlist of peptides was collated and used to determine the proteins thatwere detected in that lysate. That process was repeated for multipleLiquid Tissue lysates, and the very large list of peptides was collatedinto a single dataset. That type of dataset can be considered torepresent the peptides that can be detected in the type of biologicalsample that was analyzed (after protease digestion), and specifically ina Liquid Tissue lysate of the biological sample, and thus includes thepeptides for specific proteins, such as for example the AXL protein.

In one embodiment, the AXL tryptic peptides identified as useful in thedetermination of absolute or relative amounts of the AXL protein includeone or more, two or more, three or more, or four or more of the peptidesof SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ

ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:9, and SEQ ID NO:10, each of which are listed in Table 1. Each ofthose peptides was detected by mass spectrometry in Liquid Tissuelysates prepared from formalin fixed, paraffin embedded tissue. Thus,each peptide is a candidate for use in developing a quantitative SRM/MRMassay for the AXL protein in human biological samples, includingdirectly in formalin fixed patient tissue.

TABLE 1 Peptide Peptide Sequence SEQ ID NO: 1 GLTGTLR SEQ ID NO: 2TATITVLPQQPR SEQ ID NO: 3 LGSLHPHTPYHIR SEQ ID NO: 4 APLQGTLLGYRSEQ ID NO: 5 YGEVFEPTVER SEQ ID NO: 6 TTEATLNSLGISEELK SEQ ID NO: 7HGDLHSFLLYSR SEQ ID NO: 8 IYNGDYYR SEQ ID NO: 9 GQTPYPGVENSELYDYLRSEQ ID NO: 10 EDLENTLK

The AXL tryptic peptides listed in Table 1 include those detected frommultiple Liquid Tissue lysates of multiple different formalin fixedtissues of different human organs including lung, colon, and breast.Each of those peptides is considered useful for quantitative SRM/MRMassay of the AXL protein in formalin fixed tissue. Further data analysisof these experiments indicated no preference is observed for anyspecific peptides from any specific organ site. Thus, each of thesepeptides is believed to be suitable for conducting SRM/MRM assays of theAXL protein on a Liquid Tissue lysate from any formalin fixed tissueoriginating from any biological sample or from any organ site in thebody.

An important consideration when conducting an SRM/MRM assay is the typeof instrument that may be employed in the analysis of the peptides.Although SRM/MRM assays can be developed and performed on any type ofmass spectrometer, including a MALDI, ion trap, or triple quadrupole,the most advantageous instrument platform for an SRM/MRM assay is oftenconsidered to be a triple quadrupole instrument platform. That type of amass spectrometer may be considered to be the most suitable instrumentfor analyzing a single isolated target peptide within a very complexprotein lysate that may consist of hundreds of thousands to millions ofindividual peptides from all the proteins contained within a cell.

In order to most efficiently implement an SRM/MRM assay for each peptidederived from the AXL protein it is desirable to utilize information inaddition to the peptide sequence in the analysis. That additionalinformation may be used in directing and instructing the massspectrometer (e.g. a triple quadrupole mass spectrometer) to perform thecorrect and focused analysis of specific targeted peptide(s), such thatthe assay may be effectively performed.

The additional information about target peptides in general, and aboutspecific AXL peptides, may include one or more of the mono isotopic massof the peptide, its precursor charge state, the precursor m/z value, them/z transition ions, and the ion type of each transition ion. Additionalpeptide information that may be used to develop an SRM/MRM assay for theAXL protein is shown by example for three (3) of the AXL peptides fromthe list in Table 1 and is shown in Table 2. Similar additionalinformation described for the three (3) AXL peptides shown by example inTable 2 may be prepared, obtained, and applied to the analysis of theother peptides contained in Table 1.

TABLE 2 Mono Precursor Pre- Trans- Peptide Isotopic Charge cursor itionIon SEQ ID sequence Mass State m/z m/z Type SEQ ID TATITVLPQQPR1324.5435 2 662.883  625.341  y5 NO: 2 2 662.883  738.425  y6 2 662.883 837.494  y7 2 662.883  938.541  y8 SEQ ID APLQGTLLGYR 1188.3926 2 594.84  559.322 y10 NO: 4 2  594.84  779.441  y7 2  594.84  907.499  y82  594.84 1020.583  y9 SEQ ID YGEVFEPTVER 1325.4408 2 663.322  350.134 b3 NO: 5 2 663.322 601.33  y5 2 663.322  730.372  y6 2 663.322  877.441 y7

The method described below was used to: 1) identify candidate peptidesfrom the AXL protein that can be used for a mass spectrometry-basedSRM/MRM assay for the AXL protein, 2) develop an individual SRM/MRMassay, or assays, for target peptides from the AXL protein in order tocorrelate and 3) apply quantitative assays to cancer diagnosis and/orchoice of optimal therapy.

Assay Method

-   1. Identification of SRM/MRM candidate fragment peptides for the AXL    protein    -   a. Prepare a Liquid Tissue protein lysate from a formalin fixed        biological sample using a protease or proteases, (that may or        may not include trypsin), to digest proteins    -   b. Analyze all protein fragments in the Liquid Tissue lysate on        an ion trap tandem mass spectrometer and identify all fragment        peptides from the AXL protein, where individual fragment        peptides do not contain any peptide modifications such as        phosphorylations or glycosylations    -   c. Analyze all protein fragments in the Liquid Tissue lysate on        an ion trap tandem mass spectrometer and identify all fragment        peptides from the AXL protein that carry peptide modifications        such as for example phosphorylated or glycosylated residues    -   d. All peptides generated by a specific digestion method from        the entire, full length AXL protein potentially can be measured,        but preferred peptides used for development of the SRM/MRM assay        are those that are identified by mass spectrometry directly in a        complex Liquid Tissue protein lysate prepared from a formalin        fixed biological sample    -   e. Peptides that are specifically modified (phosphorylated,        glycosylated, etc.) in patient tissue and which ionize, and thus        detected, in a mass spectrometer when analyzing a Liquid Tissue        lysate from a formalin fixed biological sample are identified as        candidate peptides for assaying peptide modifications of the AXL        protein-   2. Mass Spectrometry Assay for Fragment Peptides from the AXL    Protein    -   a. SRM/MRM assay on a triple quadrupole mass spectrometer for        individual fragment peptides identified in a Liquid Tissue        lysate is applied to peptides from the AXL protein        -   i. Determine optimal retention time for a fragment peptide            for optimal chromatography conditions including but not            limited to gel electrophoresis, liquid chromatography,            capillary electrophoresis, nano-reversed phase liquid            chromatography, high performance liquid chromatography, or            reverse phase high performance liquid chromatography        -   ii. Determine the mono isotopic mass of the peptide, the            precursor charge state for each peptide, the precursor m/z            value for each peptide, the m/z transition ions for each            peptide, and the ion type of each transition ion for each            fragment peptide in order to develop an SRM/MRM assay for            each peptide.        -   iii. SRM/MRM assay can then be conducted using the            information from (i) and (ii) on a triple quadrupole mass            spectrometer where each peptide has a characteristic and            unique SRM/MRM signature peak that precisely defines the            unique SRM/MRM assay as performed on a triple quadrupole            mass spectrometer    -   b. Perform SRM/MRM analysis so that the amount of the fragment        peptide of the AXL protein that is detected, as a function of        the unique SRM/MRM signature peak area from an SRM/MRM mass        spectrometry analysis, can indicate both the relative and        absolute amount of the protein in a particular protein lysate.        -   i. Relative quantitation may be achieved by:            -   1. Determining increased or decreased presence of the                AXL protein by comparing the SRM/MRM signature peak area                from a given AXL peptide detected in a Liquid Tissue                lysate from one formalin fixed biological sample to the                same SRM/MRM signature peak area of the same AXL                fragment peptide in at least a second, third, fourth or                more Liquid Tissue lysates from least a second, third,                fourth or more formalin fixed biological samples            -   2. Determining increased or decreased presence of the                AXL protein by comparing the SRM/MRM signature peak area                from a given AXL peptide detected in a Liquid Tissue                lysate from one formalin fixed biological sample to                SRM/MRM signature peak areas developed from fragment                peptides from other proteins, in other samples derived                from different and separate biological sources, where                the SRM/MRM signature peak area comparison between the 2                samples for a peptide fragment are normalized to amount                of protein analyzed in each sample.            -   3. Determining increased or decreased presence of the                AXL protein by comparing the SRM/MRM signature peak area                for a given AXL peptide to the SRM/MRM signature peak                areas from other fragment peptides derived from                different proteins within the same Liquid Tissue lysate                from the formalin fixed biological sample in order to                normalize changing levels of AXL protein to levels of                other proteins that do not change their levels of                expression under various cellular conditions.            -   4. These assays can be applied to both unmodified                fragment peptides and for modified fragment peptides of                the AXL protein, where the modifications include but are                not limited to phosphorylation and/or glycosylation, and                where the relative levels of modified peptides are                determined in the same manner as determining relative                amounts of unmodified peptides.        -   ii. Absolute quantitation of a given peptide may be achieved            by comparing the SRM/MRM signature peak area for a given            fragment peptide from the AXL protein in an individual            biological sample to the SRM/MRM signature peak area of an            internal fragment peptide standard spiked into the protein            lysate from the biological sample            -   1. The internal standard is a labeled synthetic version                of the fragment peptide from the AXL protein that is                being interrogated. This standard is spiked into a                sample in known amounts, and the SRM/MRM signature peak                area can be determined for both the internal fragment                peptide standard and the native fragment peptide in the                biological sample separately, followed by comparison of                both peak areas            -   2. This can be applied to unmodified fragment peptides                and modified fragment peptides, where the modifications                include but are not limited to phosphorylation and/or                glycosylation, and where the absolute levels of modified                peptides can be determined in the same manner as                determining absolute levels of unmodified peptides.-   3. Apply Fragment Peptide Quantitation to Cancer Diagnosis and    Treatment    -   a. Perform relative and/or absolute quantitation of fragment        peptide levels of the

AXL protein and demonstrate that the previously-determined association,as well understood in the field of cancer, of AXL protein expression tothe stage/grade/status of cancer in patient tumor tissue is confirmed

-   -   b. Perform relative and/or absolute quantitation of fragment        peptide levels of the AXL protein and demonstrate correlation        with clinical outcomes from different treatment strategies,        wherein this correlation has already been demonstrated in the        field or can be demonstrated in the future through correlation        studies across cohorts of patients and tissue from those        patients. Once either previously established correlations or        correlations derived in the future are confirmed by this assay        then the assay method can be used to determine optimal treatment        strategy

Specific and unique characteristics about specific AXL peptides weredeveloped by analysis of all AXL peptides on both an ion trap and triplequadrupole mass spectrometers. That information includes themonoisotopic mass of the peptide, its precursor charge state, theprecursor m/z value, the transition m/z values of the precursor, and theion types of each of the identified transitions. That information mustbe determined experimentally for each and every candidate SRM/MRMpeptide directly in Liquid Tissue lysates from formalin fixedsamples/tissue; because, interestingly, not all peptides from the AXLprotein can be detected in such lysates using SRM/MRM as describedherein, indicating that AXL peptides not detected cannot be consideredcandidate peptides for developing an SRM/MRM assay for use inquantitating peptides/proteins directly in Liquid Tissue lysates fromformalin fixed samples/tissue.

A particular SRM/MRM assay for a specific AXL peptide is performed on atriple quadrupole mass spectrometer. An experimental sample analyzed bya particular AXL SRM/MRM assay is for example a Liquid Tissue proteinlysate prepared from a tissue that had been formalin fixed and paraffinembedded. Data from such as assay indicates the presence of the uniqueSRM/MRM signature peak for this AXL peptide in the formalin fixedsample.

Specific transition ion characteristics for this peptide are used toquantitatively measure a particular AXL peptide in formalin fixedbiological samples. These data indicate absolute amounts of this AXLpeptide as a function of molar amount of the peptide per microgram ofprotein lysate analyzed. Assessment of AXL protein levels in tissuesbased on analysis of formalin fixed patient-derived tissue can providediagnostic, prognostic, and therapeutically-relevant information abouteach particular patient. In one embodiment, this disclosure describes amethod for measuring the level of the tyrosine-protein kinase receptorUFO protein (AXL) in a biological sample, comprising detecting and/orquantifying the amount of one or more modified or unmodified AXLfragment peptides in a protein digest prepared from said biologicalsample using mass spectrometry; and calculating the level of modified orunmodified AXL protein in said sample; and wherein said level is arelative level or an absolute level. In a related embodiment,quantifying one or more AXL fragment peptides comprises determining theamount of the each of the AXL fragment peptides in a biological sampleby comparison to an added internal standard peptide of known amount,wherein each of the AXL fragment peptides in the biological sample iscompared to an internal standard peptide having the same amino acidsequence. In some embodiments the internal standard is an isotopicallylabeled internal standard peptide comprises one or more heavy stableisotopes selected from ¹⁸O, ¹⁷O, ³⁴S, ¹⁵N, ¹³C, ²H or combinationsthereof.

The method for measuring the level of the AXL protein in a biologicalsample described herein (or fragment peptides as surrogates thereof) maybe used as a diagnostic indicator of cancer in a patient or subject. Inone embodiment, the results from measurements of the level of the AXLprotein may be employed to determine the diagnostic stage/grade/statusof a cancer by correlating (e.g., comparing) the level of AXL proteinfound in a tissue with the level of that protein found in normal and/orcancerous or precancerous tissues.

Because both nucleic acids and protein can be analyzed from the sameLiquid Tissue™ biomolecular preparation it is possible to generateadditional information about disease diagnosis and drug treatmentdecisions from the nucleic acids in same sample upon which proteins wereanalyzed. For example, if the AXL protein is expressed by certain cellsat increased levels, when assayed by SRM the data can provideinformation about the state of the cells and their potential foruncontrolled growth, potential drug resistance and the development ofcancers can be obtained. At the same time, information about the statusof the AXL genes and/or the nucleic acids and proteins they encode(e.g., mRNA molecules and their expression levels or splice variations)can be obtained from nucleic acids present in the same Liquid Tissue™biomolecular preparation can be assessed simultaneously to the SRManalysis of the AXL protein. Any gene and/or nucleic acid not from theAXL and which is present in the same biomolecular preparation can beassessed simultaneously to the SRM analysis of the AXL protein. In oneembodiment, information about the AXL protein and/or one, two, three,four or more additional proteins may be assessed by examining thenucleic acids encoding those proteins. Those nucleic acids can beexamined, for example, by one or more, two or more, or three or more of:sequencing methods, polymerase chain reaction methods, restrictionfragment polymorphism analysis, identification of deletions, insertions,and/or determinations of the presence of mutations, including but notlimited to, single base pair polymorphisms, transitions, transversions,or combinations thereof.

1. A method for measuring the level of the tyrosine-protein kinasereceptor UFO protein (AXL) in a human biological sample offormalin-fixed tissue, comprising detecting and/or quantifying theamount of one or more modified or unmodified AXL fragment peptides in aprotein digest prepared from said biological sample using massspectrometry; and calculating the level of modified or unmodified AXLprotein in said sample; and wherein said level is a relative level or anabsolute level.
 2. The method of claim 1, further comprising the step offractionating said protein digest prior to detecting and/or quantifyingthe amount of one or more modified or unmodified AXL fragment peptides.3. The method of claim 2, wherein said fractionating step is selectedfrom the group consisting of liquid chromatography, nano-reversed phaseliquid chromatography, high performance liquid chromatography, orreverse phase high performance liquid chromatography.
 4. (canceled) 5.The method of claim 1, wherein said protein digest comprises a proteasedigest.
 6. The method of claim 5, wherein said protein digest comprisesa trypsin digest.
 7. (canceled)
 8. The method of claim 1, wherein themode of mass spectrometry used is Selected Reaction Monitoring (SRM),Multiple Reaction Monitoring (MRM), and/or multiple Selected ReactionMonitoring (mSRM).
 9. The method of claim 1, wherein the AXL fragmentpeptide comprises an amino acid sequence as set forth as SEQ ID NO:1,SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10. 10-11. (canceled) 12.The method of claim 1, wherein the tissue is paraffin embedded tissue.13. The method of claim 1, wherein the tissue is obtained from a tumor.14-15. (canceled)
 16. The method of claim 1, further comprisingquantifying a modified or unmodified AXL fragment peptide.
 17. Themethod of claim 16, wherein quantifying the AXL fragment peptidecomprises comparing an amount of one or more AXL fragment peptidescomprising an amino acid sequence of about 8 to about 45 amino acidresidues of AXL as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:9, and SEQ ID NO:10 in one biological sample to the amount of thesame AXL fragment peptide in a different and separate biological sample.18. The method of claim 17, wherein quantifying one or more AXL fragmentpeptides comprises determining the amount of the each of the AXLfragment peptides in a biological sample by comparison to an addedinternal standard peptide of known amount, wherein each of the AXLfragment peptides in the biological sample is compared to an internalstandard peptide having the same amino acid sequence.
 19. The method ofclaim 18, wherein the internal standard peptide is an isotopicallylabeled peptide.
 20. The method of claim 19, wherein the isotopicallylabeled internal standard peptide comprises one or more heavy stableisotopes selected from ¹⁸O, ¹⁷O, ³⁴S, ¹⁵N, ¹³C, ²H or combinationsthereof.
 21. The method of claim 1, wherein detecting and/or quantifyingthe amount of one or more modified or unmodified AXL fragment peptidesin the protein digest indicates the presence of modified or unmodifiedAXL protein and an association with cancer in the subject.
 22. Themethod of claim 21, further comprising correlating the results of saiddetecting and/or quantifying the amount of one or more modified orunmodified AXL fragment peptides, or the level of said AXL protein tothe diagnostic stage/grade/status of the cancer.
 23. The method of claim22, wherein correlating the results of said detecting and/or quantifyingthe amount of one or more modified or unmodified AXL fragment peptides,or the level of said AXL protein to the diagnostic stage/grade/status ofthe cancer is combined with detecting and/or quantifying the amount ofother proteins or peptides from other proteins in a multiplex format toprovide additional information about the diagnostic stage/grade/statusof the cancer.
 24. The method of claim 1, further comprising selectingfor the subject from which said biological sample was obtained atreatment based on the presence, absence, or amount of one or more AXLfragment peptides or the level of AXL protein.
 25. The method of claim1, further comprising administering to the patient from which saidbiological sample was obtained a therapeutically effective amount of atherapeutic agent, wherein the therapeutic agent and/or amount of thetherapeutic agent administered is based upon amount of one or moremodified or unmodified AXL fragment peptides or the level of AXLprotein.
 26. The method of claim 24, wherein therapeutic agents bind theAXL protein and/or inhibit its biological activity.
 27. The method ofclaim 26, wherein the therapeutic agent is selected to specificallytarget AXL-expressing cancer cells.
 28. (canceled)